Prion diseases are fatal neurodegenerative diseases that result from the accumulation of a misfolded isoform of the prion protein (PrPSc). Up to 15% of cases result from an autosomal dominant allelic mutation of the prion protein gene (PRNP). The Ala117Val mutation of PRNP is linked to Gerstmann-Strussler-Scheinker disease (GSS), a genetic prion disease characterized by progressive ataxia, dementia, and prominent PrP amyloid plaque deposits within the brain. We constructed a transgenic mouse line that expresses the mouse homolog of human PrP-A117V, designated Tg(PrP-A116V). These mice reproduce all the major features of GSS, including progressive ataxia and PrP amyloid deposits within the cerebellum and hippocampus. Recent work suggests RNA inhibition (RNAi) is a promising therapeutic approach for neurodegenerative disease, although the focus has been largely confined to knock down of wild type (wt) genes. However, in contrast to individuals affected by sporadic prion disease, carriers of PRNP mutations can be identified far in advance of the predicted onset of disease, making them ideal candidates for preventative therapies that can be administered long before significant neuronal death has occurred. Since the effect of long-term reduction of wt PrP in a healthy adult is not known, the ideal therapy for genetic prion disease should act to selectively knock down the mutated gene without affecting the normal allele. As proof of concept to develop allele-specific RNAi as a therapy for genetic prion disease, we will design and test siRNAs that selectively knock down PrP-A116V expression in vitro and in vivo, using our Tg(PrP-A116V) mice. Initial studies will design and test several siRNAs active against selected PrP mutations, with a special focus on A116V, using cell lines stably expressing mutant or wt PrP, to optimize allele selectivity. The siRNA sequence with the greatest selectivity for PrP-A116V will be packaged in a lentiviral vector as shRNA and delivered to the cerebellum in Tg(PrP- A116V/wt-PrP) heterozygous mice, to confirm efficient neuronal uptake and selective reduction of PrP-A116V expression. Once efficacy is confirmed, the same viral vector containing shRNA or control shRNA, will be injected into cerebellum of heterozygous mice and the resultant effect on PrP expression, disease onset, clinical symptoms, and histopathologic features of disease, will be assessed using quantitative and semi- quantitative measures. A significant delay in disease onset and a reduction of histopathologic features at specific disease intervals will provide strong support for allele-specific knockdown as a potential therapy in human genetic prion disease. Moreover, it will lay the foundation for the development of several allele-specific shRNAs for other PRNP mutations and, potentially, other genetic diseases. PUBLIC HEALTH RELEVANCE: Fifteen percent of prion disease cases are linked to a mutation of the prion protein gene (PRNP) that predisposes the prion protein to acquire a misfolded conformation, aggregate, and ultimately result in neurological disease. We have generated a transgenic mouse that displays all the characteristic clinical and pathological features associated with one form of genetic prion disease, known as Gerstmann-Strussler-Scheinker disease (GSS). In an effort to develop a therapy for these untreatable diseases, we will determine whether a technology known as RNA inhibition can be developed to selectively inhibit expression of only the mutated PRNP gene, while sparing the normal PRNP gene, and whether this technique can prevent or significantly mitigate disease in our transgenic mice.